DNA is continuously subjected to endogenous or exogenous attacks that result in the formation of base or sugar lesions.
The lesions include:                lesions of purine or pyrimidine bases: oxidative lesions induced by the cellular metabolism and by photosensitization; lesions through the formation of chemical adducts, which result from the harmful action of many genotoxic agents, such as polycyclic hydrocarbons, contained in combustion products; lesions through the formation of metheno-bases or of etheno-bases;        lesions of the structure of the DNA double helix: formation of intrastrand (between two adjacent bases of the same strand) or interstrand (between two bases located on the homologous strands) bridges, generally caused by ultraviolet radiation (formation of bridges between pyrimidines, which become dimeric) or bifunctional antitumor agents, such as cisplatin and intercalating agents, which form stable covalent bonds between the bases carried by opposite strands;        spontaneous lesions, due to the fact that DNA is a partially unstable molecule: spontaneous deaminations or depurinations;        lesions through single-stranded or double-stranded breakage: produced by agents such as ionizing radiation and through the action of free radicals;        sugar lesions: the destruction of a deoxyribose results in breakage of phosphodiester bonds in the damaged site, followed by strand breakage.        
The diversity of the induced lesions is illustrated by analyzing the stable photoproducts detected following UVC irradiation: alongside the pyrimidine dimers due to the formation of a cyclobutane ring, pyrimidine (6-4) pyrimidones form between two adjacent pyrimidines. The relative proportion of the pyrimidine dimers and (6-4) products ranges from 10 to 4 for 1. Their respective effectiveness in the lethal effect and in the mutagenic effect of ultraviolet radiation is also different: the dimers have a greater cytotoxic role than the (6-4) products, whereas the reverse is true for the mutagenic effect. Similarly, ionizing radiation (γ-rays from cobalt 60, for example) simultaneously produces single-stranded or double-stranded breakages (approximately in the ratio of 9 to 1), many base addition products, base losses and, at high doses, bridges between DNA and adjacent proteins (chromosomal, for example). On average, one strand breakage is counted per modified base. The predominant role of double-stranded breakage in the cytotoxic effect of radiation is accompanied by a mutagenic effect due to base alterations.
These various types of lesions can be created on isolated DNA. For example, (6-4) photoproduct-type lesions and cyclobutane-type pyrimidine dimers are induced by UVC irradiation (Hoeijmaker et al., Mutation Res., 1990, 236, 223-238); oxidative-type lesions are induced by Fenton's reaction in the presence of hydrogen peroxide and iron (Elliot et al., Free Rad. Biol. Med., 2000, 1438-1446). Another means of preparing modified DNA consists in manipulating plasmids by means of molecular biology techniques and inserting therein an oligonucleotide obtained by chemical synthesis and containing a lesion of interest (Biade et al., J. Biol. Chem., 1997, 273, 898-902).
All living organisms have DNA repair systems intended to maintain the integrity of their genome.
Among these repair systems, two have the function of eliminating modified bases from the DNA: they are the base excision repair (BER) system and the nucleotide excision repair (NER) system:                the BER system is more specifically dedicated to the repair of small lesions in DNA, such as oxidative damage, abasic sites, base fragmentations, base methylations, etheno-bases, etc.        the NER system takes care of bulky lesions that induce distortion of the DNA double helix, such as acetylaminofluorine-DNA, cisplatin-DNA and psoralen-DNA adducts, dimers derived from UVB and UVC irradiation of DNA, covalent lesions formed between a DNA base and another molecule, etc. (Sancar et al., Annu. Rev. Genetics, 1995, 29, 69-105).        
These various repair systems have common characteristics, and in particular the following steps:                recognition of the lesion(s) by proteins belonging to the repair system,        excision of the lesion and, optionally, of the adjacent nucleotides,        resynthesis of the missing nucleotides by polymerases in the medium,        the repair generally ends with ligation of the neoformed strand with the existing DNA strand.        
In all these cases, this process comprises the elimination of the modified nucleotide and the incorporation, as a replacement in the DNA chain, of at least one nucleotide triphosphate present in the repair medium.
It should, however, be noted that repair systems, especially in eukaryotes, are very complex and many variants of this simplistic configuration exist (overall repair, repair associated with the DNA transcription, repair associated with DNA replication, etc.). Some proteins are involved in several repair systems simultaneously, others are specific for a single system, some can be induced by cellular or external factors, others have a ubiquitous and constant expression.
For the remainder of the description:
The term “substrate” refers to any DNA that may undergo a repair reaction in the presence of cell extracts and, by extension, the DNA lesions.
The term “biological medium” or “cell extract” refers to a purified by unpurified biological preparation that may contain at least one enzyme activity related to DNA repair.
A lesion can generally be associated with the specific proteins responsible for its repair in the DNA. Differences exist according to species: in prokaryotes such as Escherichia coli, the enzymes are less specific, whereas in humans, a much stricter lesion-specific repair enzyme association is observed, especially in the BER system. For example, Lindahl and Wood (Science, 1999, 286, 1897-1905) describe the BER system enzymes that are the most important in humans and also the lesions that are associated therewith. For example, in humans, the OGG1 protein, which is a glycosylase belonging to the BER system, is associated with the repair of 8-oxo-2′-deoxyguanosine. In Escherichia coli, formamidopyrimidine-DNA N-glycosylase repairs this same lesion, but more generally also oxidized purine bases (Seeberg et al., TIBS, 1995, 20, 391-397). The human protein ANPG is the equivalent of the bacterial protein AlkA. These enzymes do not, however, have the same affinities for their substrates and have different excision rate constants (Laval et al., Mut. Res., 1998, 402, 93-102). More than about forty different lesions that are taken care of by the BER system can have considerable and negative biological consequences if they are not repaired. It is considered that the enzymes responsible for their repair have a considerable antitumor role. It can be seen that precise knowledge of their substrate specificities is very important.
Cellular repair capacity assays have been developed and can be classified in two categories: in vitro assays that require the use of active cell extracts, and in vivo or semi in vivo systems carried out on live cells.
I. Methods Based on Measuring Excision/Resynthesis Activity
A. Most of the in vitro assays have been developed on the basis of the experiments described by Wood et al. (Cell, 1988, 53, 97-106 and Biochemistry, 1989, 26, 8287-8292), which assess the excision/resynthesis step of the repair.
More precisely, this assay comprises the use of a plasmid DNA into which lesions are introduced (by UV irradiation: formation of pyrimidine dimers, bridges; through the action of DNase I: single-stranded cleavage or breakage); the DNA thus modified is incubated at 30° C. in the presence of a repair preparation comprising at least: the cell extract to be assessed, a nucleotide triphosphate labeled in the alpha-position with 32P and ATP. The enzymes contained in the extract incise the plasmid DNA and eliminate the lesions. DNA is synthesized de novo by replacement of the eliminated nucleotides. The radioactive nucleotide introduced into the medium is incorporated into the DNA during the synthesis. After isolation of the repaired plasmid by agarose gel electrophoresis, the amount of radioactivity incorporated, which is proportional to the rate of repair of the substrate, is measured. The method of preparing the cell extract and the reaction conditions influence the quality of the repair. In particular, it appears that the best repair yield is obtained with whole cell extracts of the type of those used for in vitro transcriptions, whereas cytosolic extracts of the type of those used for promoting plasmid replication from an SV40 origin, and also other crude cell extracts, exhibit nuclease activity, which does not allow correct interpretation of the repair.
Under the conditions established by Wood et al., the specificity of the reaction as regards the irradiated DNA is greater in the presence of a KCl concentration of the order of 40-100 mM. In addition, the irradiated DNA replication, which takes place during repair, is highly dependent on the presence of ATP and of an ATP-regenerating system (phosphocreatine+creatine phosphokinase), with a view to maintaining a constant level of ATP, the maintaining of this level being more specifically associated with the incision step of the repair. Such a dependency is not, for example, encountered in cases of strand breakage repair. A control sample, consisting of the same plasmid that has not been modified, is used simultaneously in the reaction mixtures.
The assay from Wood et al. requires the use of radioactive labels, which imposes restrictions that limit the implementation of this method in routine assays; in addition, this assay does not have sufficient qualities of simplicity and of practicality for it to be used routinely.
The method of Wood et al. has been proposed in assays characterizing extracts originating from cells established from patients suffering from xeroderma pigmentosum (Satoh et al., Proc. Natl. Acad. Sci. USA, 1993, 90, 6335-6339; Jones et al., Nucl. Acids Res., 1992, 20, 991-995; Robins et al., EMBO J., 1991, 10, 3913-3921). Xeroderma pigmentosum is a multigenic, multiallelic, autosomal recessive disease. Cells originating from patients suffering from this disease are very sensitive to ultraviolet radiation and exhibit DNA repair deficiencies. Eight genes are involved in the various complementation groups of this disease: XPA to XPG and the variant group XPV. Each group has different characteristics relating to DNA repair and in particular to the various NER subtypes. DNA lesions, whether they belong to the small lesion category or the bulky lesion category, are repaired differently, according to the complementation group.
Other repair diseases (Cockayne Syndrome, Ataxia Telangectasia) also have specific repair characteristics and have been studied by the method of Wood et al.
B. In various publications, the team of B. Saliles and P. Calsou (Biochimie 1995, 77, 796-802; Anal. Biochem. 1995, 232, 37-42) describes a method for detecting DNA lesions by carrying out excision/resynthesis reactions on a plasmid attached in wells of microplates. The plasmid is adsorbed in microplate wells and then modified, a posteriori, with chemical agents. Cell extracts are added to the wells along with digoxigenin-labeled nucleotide triphosphates. The label is incorporated into the DNA, if lesion excision has occurred, during the resynthesis step. The label is then revealed in each well by means of an antibody coupled to alkaline phosphatase. A substrate that becomes luminescent after dephosphorylation by alkaline phosphatase is added to each well. The luminescent signal, emitted in each well, is measured. It is proportional to the rate of incorporation of the label.
This method is also described in international application WO 96/28571, the inventors of which also belong to the team of B. Salles and P. Calsou, and which describes a method for qualitatively and quantitatively detecting DNA lesions, in which DNA with lesions is attached to a solid support and a composition comprising a cell extract to be tested and containing labels is brought into contact with said DNA with lesions (prior or subsequent to the attachment to said solid support). They consider that their method makes it possible to simultaneously process a large number of samples; if reference is made to the examples, the repair in the presence of a cell extract is carried out in a reaction medium of 50 μl, using an extract comprising 150 μg of proteins, 50 mM of KC, 5 mM of magnesium chloride, DTT, phosphocreatine, phosphocreatine kinase and various dNTPs, one of them being labeled with digoxigenin. The repair is obtained after incubation for 3 hours at 30° C. and the wells are washed with a washing solution comprising a phosphate buffer with a salt, to which a nonionic surfactant (Tween 20) is added in a proportion of 0.05 and 0.15% (preferred composition: 10 mM phosphate buffer, 137 mM NaCl and 0.1% Tween 20). It is specified that this assay is highly sensitive, insofar as the detection is carried out on 40 ng of DNA instead of 200 or 300 ng, in the case of an assay solution.
The assay by the team of B. Salles and P. Calsou essentially proposes modifying the plasmid after attachment to the solid support. Now, it is known that, included among the lesions created by many chemical or physical agents are chain breakages. Now, these breakages are repaired very rapidly and effectively by active cell extracts. With this assay, it is therefore impossible to differentiate between breakage repair and the repair of other lesions. Breakage repair can even mask the repair of other lesions and interfere with the signals attributed to the repair of other DNA lesions. It is therefore a system that allows the detection of an overall effect, without identifying the lesions recognized by the repair systems; in addition, the aim of this method is not to detect and quantify the activity of the proteins involved in the DNA repair, but to identify the presence of lesions on the DNA processed.
II. Methods Based on Assessing the Incision/Excision Step
Variants of the method by Wood et al. have been proposed and make it possible in particular to measure only the lesion incision activity:
A. Redaelli et al. (Terat. Carcinog. Mut., 1998, 18, 17-26) describe a method in which the plasmid is incubated directly with the extract without the nucleotide triphosphates. Cleavages in the supercoiled plasmid bring about a change in migration rate in the agarose gel during electrophoresis. The supercoiled plasmid migrates faster that the incised plasmid, due to its conformation. The bands corresponding to the various forms of the plasmid are quantified; the amount of the incised form is correlated with the activity of incision of the lesions of the plasmid, containing the extract.
More precisely, this article studies the incision action of AP-endonuclease, that occurs on an a basic site, obtained after the action of a glycosylase specific for the modification to be repaired (alkylation, hydrolytic deamination, oxidation, mismatching), by cleaving the deoxyribose phosphodiester linkage positioned 3′ or 5′ of this abasic site. In this article, the AP-endonuclease activity is more specifically studied on a crude extract of human lymphocytes. The extract (80 μl) is incubated, firstly, with an undamaged plasmid (control) and, secondly, with a depurinated plasmid. It is thus found to be possible to quantify the activity of AP-endonuclease insofar as the incision activity is dependent on the damage and sensitive to EDTA.
B. The team of P. Calsou and B. Salles (Biochem. Biophys. Res. Corm., 1994, 202, 788-795) proposes another approach for specifically measuring the activity of incision of the lesions of a plasmid. They introduce into the repair medium an inhibitor specific for eukaryotic polymerases, aphidicolin, in order to prevent resynthesis, by the endogenous polymerases, of the normal DNA fragments after the first excision step. An exogenous prokaryotic polymerase is mixed with the reaction medium in an equivalent amount for all the tubes. Differences in the results obtained thus reflect the lesion excision step and not the resynthesis of the excised DNA fragments.C. Another method based on a modification of the comet assay (gel electrophoresis, in an alkali medium, of a single cell) also makes it possible to measure incision activity. It was developed by Collins et al. (Mutagenesis, 2001, 16, 297-301). Oxidative lesions are introduced into the genomic DNA by photosensitization of HeLa cells in the presence of visible light. The cells are then incorporated into an agarose gel spread onto a microscope slide, and the cell membranes and the proteins are then eliminated by controlled lysis. The nucleoids isolated in the gel are incubated in the presence of cell extracts that are active for the first lesion incision step. The slides are then subjected to electrophoresis in an alkali medium. The presence of cleavage induces more rapid migration of the DNA than the nucleoid as a whole. The ball of DNA then has the appearance of a comet, the intact DNA being in the head and the DNA containing cleavages being in the tail of the comet. The percentage of DNA in the tail of the comet, determined by means of specialized software, correlates directly with the incision activity contained in the extracts used for the lesions under consideration. This assay has been applied to measuring activities of excision of oxidative damage in extracts originating from human lymphocytes. Compared with the method described by Redaelli et al., 1998, which measures the cleavage of plasmids, the method of Collins et al. uses the comet method to estimate strand cleavages and considers that this variant is, firstly, significantly more sensitive (detection of approximately 0.2 to 2 cleavages per 109 daltons) and, secondly, economically advantageous (savings in terms of the material used), since the volume of reaction mixture (DNA included in a gel) is only 50 μl and a sufficient amount of material to be assayed is obtained from 10 ml of blood (possibility of carrying out several incubations).D. International application WO 01/90408 describes a method for detecting and characterizing activities of proteins involved in DNA lesion repair.
More precisely, this method comprises the attachment to a solid support of at least one damaged DNA containing at least one known lesion; this damaged DNA is then subjected to the action of a repair composition containing or not containing at least one protein involved in the repair of this damaged DNA, and the determination of the activity of this protein for the repair by measuring the variation of a signal emitted by a label that attaches to or removes itself from the support during the preceding step.
This system, which is used with a damaged DNA that is in the form of an oligonucleotide of 15 to 100 bases or of a polynucleotide of 100 to 20 000 bases, thus makes it possible to obtain a more overall piece of information than the other assays, since the excision of several substrates can be monitored simultaneously.
However, this method concerns the demonstration of DNA lesion incision activities. It is thus limited to characterizing the step of excision of lesions that may be introduced into synthetic oligonucleotides. Furthermore, although it provides considerable information regarding excision activities, it is not suitable for and does not describe a precise quantification of the enzyme activities for excision/resynthesis of DNA.
Besides the drawbacks specific to each technique, reported above, these various methods also have the following drawbacks:                All the assays described above require the use of amounts of biological material and in particular of cell extracts of greater than 10 μl: the reaction volume generally used is 50 μl containing from 10 to 40 μl of extract for an amount of proteins of approximately 100 μg. The extracts take a long time to prepare, and the amount of available cells is often small, which limits the number of assays that can be carried out.        Whatever the detection method used and whether the assay is carried out in solution or on a support, all these systems described provide information regarding point-by-point repair limited to a given substrate for an aliquot fraction of extract; this is because, in the assays for determining repair capacities as proposed by Wood et al., for example, each assay is carried out individually in a tube, i.e. a reaction takes place in the presence of a given plasmid and of a given extract. For each extract to be tested, the rate of incorporation of the label into the plasmid is compared with the rate of incorporation of the label obtained in a substrate prepared in an identical manner in the presence of the control extract. The reference control extract is generally prepared from characterized cells transformed with EBV or SV40. The same is true in most of the other variants of the method by Wood et al., described above.        Since the assays are relatively laborious to carry out and require the availability of large amounts of biological material, experimenters limit the number of substrates used and the number of biological extracts tested.        The lesions introduced into the plasmids are neither measured nor quantified. The authors, using the assay developed by Wood et al., eliminate only the plasmids that have lost their supercoiled form, in order to eliminate DNA containing chain breakages. The information obtained is very partial and insufficient to precisely define and characterize the repair capacities of a given biological medium.        